Caveolin-1 is a negative regulator of endothelial nitric oxide synthase (eNOS) function. Indeed, caveolin-1 (-/-) mice exhibit enhanced endothelium-dependent relaxations in conduit vessels and exaggerated NO-dependent microvascular permeability in response to vascular endothelial growth factor (VEGF). We have shown that a cell-permeable, caveolin scaffolding domain (CSD) attenuates endothelium-dependent relaxations, and reduces vascular permeability in vivo. With this background in mind, we hypothesize that understanding the atomic interactions between eNOS and caveolin-1 will permit molecular dissection of the physiological and pathophysiological roles of caveolin-1 as a negative regulator of eNOS. To examine the regulation of this important interaction in more detail, we will: Aim 1. Define the molecular interactions of eNOS with caveolin-1 at the atomic level. We will refine our structure of the CSD bound to eNOS and test the structural model using biophysical, biochemical and cell biological approaches. We will mutate key amino acid residues at the interface of eNOS and caveolin-1 and examine their effects on NOS activity and NO release; Aim 2. Develop new cell-permeable peptides fused to the CSD based on structural information and test their effects in vivo. In preliminary results, we show a cell permeable version of CSD stereospecifically blocks endothelium-dependent relaxations, acute changes in vascular permeability and tumor permeability and progression in vivo. Moreover, we have isolated a small fragment of CSD that exerts biological activity in cells; and Aim 3. Examine vascular function in transgenic mice that express mutant eNOS or caveolin-1. Based on structural information, we will define generate transgenic mice expressing wild-type or mutant CSD to directly test the molecular nature of this interaction. We will monitor systemic hemodynamics, vascular function, permeability, and tumor progression as physiological endpoints in these mice. [unreadable] [unreadable]